One realization that has come from comparing multiple bacterial genome sequences, including multiple isolates from the same species, is that gene transfer is an important force in bacterial genome evolution. In the laboratory gene transfer is essential for the study of bacteria and for learning more about all living organisms. Three processes in bacteria can broadly define the transfer of DNA: transformation, transduction, and conjugation. This chapter focuses on the many genetic tools available to manipulate the genetic content of Escherichia coli. A DNA molecule that does not have its own origin of replication must integrate into either the host chromosome or another autonomously replicating element such as an endogenous plasmid. In E. coli a modified derivative of the bacteriophage T4 offers some advantages for transduction in that it packages twice as much DNA as P1 and also is less sensitive to capsules found on many pathogenic strains of E. coli. Transformation of bacteria by use of either naturally competent organisms, the process of electroporation, or chemical competency relies on the direct uptake of DNA by bacteria. Conjugation can be used as a tool to deliver plasmids that are capable of being stably maintained in the target host or as a tool to deliver “suicide vectors” that cannot replicate in the target host.

Genetic information can be integrated into the chromosome of a bacterium using a single crossover from a circular DNA construct that cannot replicate in the host. (a) A single open reading frame (thick line) to be inactivated is shown with its promoter (solid arrow) and RNA transcript (dashed line with arrow). (b) A plasmid (circle) that cannot replicate in the strain can be maintained only by integrating into the chromosome using homology provided on the plasmid indicated with an “X.” Three arbitrary positions are indicated with numbers to show the orientation. (c) Integration of the circular DNA substrate disrupts and fuses the plasmid-borne genes behind the target gene promoter. Depending on the construct, the transcript may be terminated within the integrated DNA segment. Promoters within the plasmid could also activate adjacent genes in the chromosome.

10.1128/9781555817497/fig31-1_thmb.gif

10.1128/9781555817497/fig31-1.gif

FIGURE 1

Genetic information can be integrated into the chromosome of a bacterium using a single crossover from a circular DNA construct that cannot replicate in the host. (a) A single open reading frame (thick line) to be inactivated is shown with its promoter (solid arrow) and RNA transcript (dashed line with arrow). (b) A plasmid (circle) that cannot replicate in the strain can be maintained only by integrating into the chromosome using homology provided on the plasmid indicated with an “X.” Three arbitrary positions are indicated with numbers to show the orientation. (c) Integration of the circular DNA substrate disrupts and fuses the plasmid-borne genes behind the target gene promoter. Depending on the construct, the transcript may be terminated within the integrated DNA segment. Promoters within the plasmid could also activate adjacent genes in the chromosome.

Genetic information can be integrated into the chromosome of a bacterium using a double-crossover event from a circular DNA construct that cannot replicate in the host. (a) A single open reading frame (thick line) to be inactivated is shown with its promoter (solid arrow) and RNA transcript (dashed line with arrow). (b) A plasmid (oval) that cannot replicate in the strain can be maintained only by integrating into the chromosome using the homology provided on the plasmid indicated with “X's” (thick lines). Two crossover events ensure that only a portion of the circular DNA is integrated into the gene. Five arbitrary positions are indicated with numbers to show the DNA that is integrated and the orientation. A single crossover event likely occurs at a mid-step in the reaction and is not shown (Fig. 1). (c) Integration of the circular DNA substrate removes all or a portion of the gene and fuses the encoded genes behind the target gene promoter. Depending on the construct, the transcript may be terminated within the integrated DNA segment. Promoters within the plasmid could activate adjacent genes in the chromosome.

10.1128/9781555817497/fig31-2_thmb.gif

10.1128/9781555817497/fig31-2.gif

FIGURE 2

Genetic information can be integrated into the chromosome of a bacterium using a double-crossover event from a circular DNA construct that cannot replicate in the host. (a) A single open reading frame (thick line) to be inactivated is shown with its promoter (solid arrow) and RNA transcript (dashed line with arrow). (b) A plasmid (oval) that cannot replicate in the strain can be maintained only by integrating into the chromosome using the homology provided on the plasmid indicated with “X's” (thick lines). Two crossover events ensure that only a portion of the circular DNA is integrated into the gene. Five arbitrary positions are indicated with numbers to show the DNA that is integrated and the orientation. A single crossover event likely occurs at a mid-step in the reaction and is not shown (Fig. 1). (c) Integration of the circular DNA substrate removes all or a portion of the gene and fuses the encoded genes behind the target gene promoter. Depending on the construct, the transcript may be terminated within the integrated DNA segment. Promoters within the plasmid could activate adjacent genes in the chromosome.

Genetic information can be integrated into the chromosome of a bacterium using a double-crossover event from a linear DNA construct. (a) A single open reading frame (thick line) to be inactivated is shown with its promoter (solid arrow) and RNA transcript (dashed line with arrow). (b) Two crossover events must occur for the linear DNA to be integrated into the gene when selecting for gene products indicated by 1 or 2. Homology provided on the fragment is indicated with “X's.” (c) Integration of the DNA substrate removes all or a portion of the gene and fuses the genes carried behind the target gene promoter. Depending on the construct, the transcript may be terminated within the integrated DNA segment. Promoters within the integrated DNA segment could activate adjacent genes in the chromosome.

10.1128/9781555817497/fig31-3_thmb.gif

10.1128/9781555817497/fig31-3.gif

FIGURE 3

Genetic information can be integrated into the chromosome of a bacterium using a double-crossover event from a linear DNA construct. (a) A single open reading frame (thick line) to be inactivated is shown with its promoter (solid arrow) and RNA transcript (dashed line with arrow). (b) Two crossover events must occur for the linear DNA to be integrated into the gene when selecting for gene products indicated by 1 or 2. Homology provided on the fragment is indicated with “X's.” (c) Integration of the DNA substrate removes all or a portion of the gene and fuses the genes carried behind the target gene promoter. Depending on the construct, the transcript may be terminated within the integrated DNA segment. Promoters within the integrated DNA segment could activate adjacent genes in the chromosome.

Recombination substrates can be made in vitro for use with λ Red recombination. The antibiotic resistance used to select for recombinants can subsequently be removed by expressing the Flp recombinase that will excise the region between the two frt sites (FRT). (a) Primers are designed to amplify from a universal antibiotic-resistant cassette with frt recombination sites using primer binding sites P1 and P2. The primers contain 5' “tails” that are complementary to the target gene at regions H1 and H2 for the subsequent crossover event. (b) The PCR-generated substrate is transformed into a cell expressing the Exo, Beta, and Gam proteins (substrate shown miniaturized). (c) Selecting for the antibiotic resistance marker crosses in the cassette with its flanking frt recombination sites using the homology at regions H1 and H2. (d) The Flp recombinase is transiently expressed and subsequently lost because replication from the pCP20 vector is temperature sensitive. The Flp recombinase efficiently catalyzes recombination between the frt recombination sites removing the antibiotic resistance-conferring cassette. See the text for details.

10.1128/9781555817497/fig31-4_thmb.gif

10.1128/9781555817497/fig31-4.gif

FIGURE 4

Recombination substrates can be made in vitro for use with λ Red recombination. The antibiotic resistance used to select for recombinants can subsequently be removed by expressing the Flp recombinase that will excise the region between the two frt sites (FRT). (a) Primers are designed to amplify from a universal antibiotic-resistant cassette with frt recombination sites using primer binding sites P1 and P2. The primers contain 5' “tails” that are complementary to the target gene at regions H1 and H2 for the subsequent crossover event. (b) The PCR-generated substrate is transformed into a cell expressing the Exo, Beta, and Gam proteins (substrate shown miniaturized). (c) Selecting for the antibiotic resistance marker crosses in the cassette with its flanking frt recombination sites using the homology at regions H1 and H2. (d) The Flp recombinase is transiently expressed and subsequently lost because replication from the pCP20 vector is temperature sensitive. The Flp recombinase efficiently catalyzes recombination between the frt recombination sites removing the antibiotic resistance-conferring cassette. See the text for details.

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